Molecular Origins of Phenotypic Changes in the Obese Microvascular Endothelium

项目概要 肥胖是动脉粥样硬化、高血压、2 型心血管疾病 (CVD) 的主要危险因素 糖尿病和中风。尽管有充分证据表明大多数形式的 CVD 中都存在 EC 功能障碍，但 肥胖导致 EC 功能障碍的机制仍知之甚少。进一步 整个脉管系统中 EC 的异质性使对 EC 功能障碍的理解变得复杂。 最近的研究表明 EC 亚型之间在大循环方面存在很大程度的差异 （主动脉）可以在短时间内通过致肥饮食进一步改变。我们的实验室和其他实验室有 研究表明，微血管系统特别容易受到肥胖引起的功能障碍的影响，并且是一个关键的预测因素 终末器官损伤。在肥胖小鼠的微脉管系统中，我们发现 NADPH 显着增加 氧化酶 1 (NOX1) 和组蛋白脱乙酰酶 9 (HDAC9)，这两个分子已被证明很重要 分别导致内皮功能障碍和表型变化。截至本提案撰写之时， 目前尚无研究阐明微循环中 EC 的异质性，更不用说 肥胖对这些人群的影响，或者 NOX1 和 HDAC9 是否在某些人群中优先表达 人口超过其他人。为了以时间和成本有效的方式进行这些实验，我们开发了 一种 AAV 方法，可将 AgRP（一种促进食欲的肽）递送至大脑，以诱导食欲亢进和 肥胖。这种新方法与内皮特异性谱系追踪小鼠和内皮特异性相结合 HDAC9 敲除小鼠将有助于测试我们的中心假设，即肥胖改变了 EC 异质性 微循环有利于表达 NOX1 的炎症 EC 表型。目标 1 将通过肥胖定义 内皮特异性谱系追踪模型，转录组和表型变化潜在的损失 肥胖小鼠微循环中的内皮功能。使用命运图谱 EC 的单细胞 RNA 测序 从瘦小鼠和肥胖小鼠中，我们将阐明表达 NOX1 的 EC 亚型并询问共表达 模式。目标 2 将检验以下假设：HDAC9 在 NOX1 和 NOX1 表达增加中具有因果作用 微血管内皮功能障碍，体重减轻逆转了 HDAC9 和 NOX1 的表达， 恢复内皮功能。我们将在体外和体内评估HDAC9是否驱动NOX1的表达 以及它的缺失是否可以改善肥胖症的内皮功能障碍。我们还将调查影响 使用新型 floxed AgRP AAV 减轻体重对微血管内皮功能的影响 AgRP 表达的目的是促进减肥。我们预计该提案的调查结果将揭示 导致肥胖症内皮功能障碍的新机制，并将提供对潜在潜力的洞察 改善心血管健康的治疗目标。

PROJECT SUMMARY Obesity is a major risk factor for cardiovascular diseases (CVD) such as atherosclerosis, hypertension, type 2 diabetes and stroke. Although it is well documented that EC dysfunction is present in most forms of CVD, the mechanisms by which obesity shapes the development of EC dysfunction remains poorly understood. Further complicating the understanding of EC dysfunction is the heterogeneity of ECs throughout the vasculature. Recent studies have demonstrated a large degree of divergence between EC subtypes in the macrocirculation (aorta) that can be further altered with obesogenic diets over a short period of time. Our lab and others have shown that the microvasculature is particularly susceptible to obesity induced dysfunction and is a key predictor of end-organ damage. In the microvasculature of obese mice, we have shown a striking increase in NADPH oxidase 1 (NOX1) and histone deacetylase 9 (HDAC9), two molecules that have been shown to be important contributors to endothelial dysfunction and phenotypic changes, respectively. As of the writing of this proposal, no studies have been conducted to elucidate the heterogeneity of EC in the microcirculation, not to mention the effect of obesity on these populations or whether or not NOX1 and HDAC9 are preferentially expressed in certain populations over others. To perform these experiments in a time and cost effective manner, we have developed an AAV approach to deliver AgRP, an orexigenic peptide, specifically to the brain to induce hyperphagia and obesity. This novel approach in conjunction with endothelial specific lineage tracing mice and endothelial specific HDAC9 knockout mice will facilitate testing of our central hypothesis that obesity alters EC heterogeneity in the microcirculation to favor inflammatory EC phenotypes expressing NOX1. Aim 1 will define, through an obese endothelial specific lineage tracing model, the transcriptomic and phenotypic changes underlying loss of endothelial function in the microcirculation of obese mice. Using single cell RNA sequencing of fate mapped ECs from lean and obese mice, we will elucidate the subtypes of ECs expressing NOX1 and interrogate co-expression patterns. Aim 2 will test the hypothesis that HDAC9 has a causal role in increased expression of NOX1 and microvascular endothelial dysfunction and that weight loss reverses the expression of HDAC9 and NOX1 and restores endothelial function. We will assess in vitro and in vivo, whether HDAC9 drives the expression of NOX1 and whether its deletion ameliorates endothelial dysfunction in obesity. We will also investigate the effects of weight loss on microvascular endothelial function using a novel floxed AgRP AAV that enables cessation of AgRP expression with the goal of promoting weight loss. We anticipate the findings of this proposal will reveal new mechanisms contributing to endothelial dysfunction in obesity and will provide insight into potential therapeutic targets to improve cardiovascular health.